HTML
-
Pigs in groups Vac/ChA, Vac/ChB, and Mock were clinically normal from the beginning of the study (0 dpv) to the end of challenge (21 dpc); this included the respiratory scores and rectal temperatures. However, the pigs from the unvaccinated groups, Unvac/ChA and Unvac/ChB, showed clear clinical symptoms. In the TJ strain challenge group, the vaccinated pigs in group Vac/ChA remained normal throughout the study, as evaluated by the clinical symptom scores (Fig. 1) and rectal temperatures (Fig. 2). However, in group Unvac/ChA (unvaccinated pigs), all pigs developed typical clinical signs of HP-PRRSV, such as severe depression and anorexia, lameness and shivering, dyspnea, skin cyanosis, and death (Li et al. 2016b). All pigs in group Unvac/ChA displayed persistently high fever (≥ 41 ℃) for 6 days from 6 dpc. The mean rectal temperature and clinical score were significantly higher in group 2 than in group Vac/ChA (P < 0.05). At 15, 18, and 19 dpc, three of five unvaccinated pigs that had been challenged in group Unvac/ChA died of acute respiratory disease (mortality rate of 60%) (Fig. 3). Unlike in pigs in the Unvac/ChB group, no clinical symptoms were observed in group Vac/ ChB. In group Unvac/ChB, four of five infected pigs presented with persistent high temperature (≥ 40 ℃) beginning 3 dpc that lasted for 7 days. However, one pig experienced persistent high fever (≥ 40 ℃) between 11 and 15 dpc, and then died 18 dpc. Clinical differences between groups Vac/ChB and Unvac/ChB were reflected in the rectal temperatures and clinical scores (Lager et al. 2014).
Figure 1. Clinical scores in vaccinated or unvaccinated pigs. Pigs were observed every day for clinical signs including depression, cough, excessive lacrimation, and rectal temperature, and they were scored daily for clinical respiratory disease severity using scores ranging from 0 (normal) to 6 (severe dyspnea and abdominal breathing). Vac/ChA: Vaccinated + Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated + Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.
Figure 2. Daily monitoring of rectal temperature of all the pigs post challenge. The clinical threshold for "fever" in the NADC30-like strain and HP-PRRSV strain was set at 40 and 41 ℃, respectively. Vac/ChA: Vaccinated + Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated + Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.
Figure 3. The survival rate for each challenged group. At 15, 18, and 19 dpc, three of the five unvaccinated pigs in group Unvac/ChA that had been challenged died of acute respiratory disease (mortality rate of 60%) and one pig died at 18 dpc in group Unvac/ChB. Vac/ChA: Vaccinated + Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated + Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.
There was no noticeable difference in clinical symptoms between Vac/ChA and Vac/ChB animals. In contrast, clear differences in clinical symptoms were observed between the Unvac/ChA and Unvac/ChB animals. The pigs in the Unvac/ChB group presented with mild clinical symptoms as evidenced by the mean rectal temperature, clinical score, and mortality rate. The results were consistent with those of a previous study (Sun et al. 2016). The pigs in the Mock group maintained normal temperatures without respiratory symptoms throughout the experiment. No bacterial pathogens were isolated at necropsy.
-
All pigs in all five groups were seronegative for PRRSVspecific antibody at 0 dpv. Pigs in the Vac/ChA and Vac/ChB groups developed antibodies that were detected using ELISA from 14 dpv onwards; in the Unvac/ChA and Unvac/ChB groups, these were detected from 6 dpc onwards (Fig. 4) The S/P ratio reached the highest level at 6 dpc, following which it gradually decreased in the vaccinated groups from the highest level at 10 dpc in the Unvac/ChA group and 14 dpc in the Unvac/ChB group. No anti-PRRSV antibodies were detected in mock group throughout the experiment. The results of virus neutralization assays showed that NAs were not detected in any groups from 0 to 21 dpc (data not shown).
Figure 4. Commercial ELISA kits were used to test the PRRSV-specific antibodies. S/P value = sample OD650 divided by negative control OD650. S/P ≥ 0.4, positive; S/P < 0.4, negative. Vac/ChA; Vaccinated +Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated +Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.
-
No virus was detected in the Vac/ChA and Vac/ChB groups (Table 1). However, the groups that were challenged (but received no vaccine) did shed virus. Three pigs in the Unvac/ChA groups shed virus for 12, 14, and 16 days, respectively from 2 dpc onwards, as against the other two pigs, which that stopped shedding virus after 10 dpc. In the Unvac/ChB group, four pigs shed virus for 4–6 days, and one pig shed virus from 8 to 16 dpc, at which point it died. Furthermore, the virus shedding rate per day in the Unvac/ChA group was higher than that in the Unvac/ChB group.
Table 1. Viremia from post-challenge pigs.
-
After all four groups were challenged with the PRRSV, pigs in the vaccinated groups continued to gain body weight until euthanized. There was no significant difference in body weight between the vaccinated-challenged groups and the mock group. However, all the pigs in the unvaccinated-challenged groups experienced weight loss to a greater extent than did pigs in the vaccinated and mock groups. The pigs in group Unvac/ChA lost significantly more body weight than those in group Unvac/ChB (Fig. 5).
Figure 5. Body weight gain of pigs in all the groups. All the pigs in the unvaccinated-challenged groups experienced weight loss compared with those in the vaccinated and mock groups. Vac/ChA: Vaccinated + Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated + Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.
-
All surviving pigs were euthanized at 21 dpc and subjected to necropsy. In the unvaccinated-challenged group, Unvac/ChA pigs exhibited severe gross lesions with consolidation, hemorrhage, firmer and heavier parenchyma in the lung tissues with hemorrhage, and swelling of the lymph nodes. This differed from observations of lighter parenchyma of the lung and fewer pathological changes in the Unvac/ChB pigs (Fig. 6). However, there were no visible gross pathological changes in the vaccinated-challenged groups, nor were there any lesions in the tissues of the mock group.
Figure 6. Macroscopic examination of the lungs (A1–E1) and lymph nodes (A2–E2). A1 and A2 (Vac/ChA group), C1 and C2 (Vac/ChB group), and E1 and E2 (mock group) indicate the normal lungs and lymph nodes. B1 indicates extensive pneumorrhagia and lung consolidation. B2: Extensive hemorrhage in the lymph nodes. D1 indicates mild lung consolidation. D2 indicates slight hemorrhage in the lymph nodes. Vac/ChA: Vaccinated + Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated + Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.
Microscopic pulmonary lesions were typical of those associated with PRRSV infection. Therefore, pathological evaluation that includes detection of the PRRSV antigen within these lesions is also critical for determining the efficacy of PRRSV vaccines (Opriessnig et al. 2007). Consistent with the macroscopic findings, the histological examination results also revealed that the Unvac/ChB pigs exhibited mild interstitial pneumonia with symptoms of epithelial proliferation and fewer fibroblasts. Furthermore, the lymph nodes of these pigs exhibited less proliferation of cortical lymphocytes. However, severe interstitial pneumonia and acute hemorrhage were observed in the Unvac/ChA pigs (Fig. 7). IHC staining results revealed different intensities of positive staining in the lung tissues of the Unvac/ChA and Unvac/ChB pigs. However, no positive staining was detected in the mock group (Fig. 7).
Figure 7. Histopathological examination of the lungs (A1–E1) and lymph nodes (A2–E2) and IHC examination of the lungs (A3–E3). A1–A3, C1–C3, and E1–E3 indicate the normal lungs and lymph nodes. B1 indicates serious bronchopneumonia. B2 indicates serious hemorrhage in the lymph nodes and lymphopenia. Positive staining was detected in B3. D1 indicates milder interstitial pneumonia and lymphoreticulosis, D2 indicates slight lymphoproliferation and milder subcortical hyperplasia. Positive staining was detected in D3. No positive staining was detected in E3. Vac/ChA: Vaccinated + Challenged TJ strain; Unvac/ChA: Challenged TJ strain; Vac/ChB: Vaccinated + Challenged HN 201605 strain; Unvac/ChB: Challenged HN 201605 strain; Mock: challenged with the same dose of PBS.